Repression of Cell Cycle–Related Proteins by Oxaliplatin but Not Cisplatin in Human Colon Cancer Cells

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Repression of cell cycle–related proteins by oxaliplatin but not cisplatin in human colon cancer cells

Carole Voland,1 Annie Bord,1 Annick Pe´leraux,1 predictive markersfor evaluating and comparing the Ge´raldine Pe´narier,1 Dominique Carrie`re,1 efficacy and molecular pharmacology of platinum drugs. Sylvaine Galie`gue,1 Esteban Cvitkovic,2 [Mol Cancer Ther 2006;5(9):2149–57] Omar Jbilo,1 and Pierre Casellas1 Introduction 1Oncology Department, Sanofi-Synthelabo Recherche, Montpellier, France and 2Hoˆpital Saint Louis (Assistance Platinum drugs are among the most widely used compounds Publique Hospitaux de Paris), Paris, France in cancer chemotherapy. The cytotoxicity of platinum derivatives is characterized by the formation of platinum adducts on DNA, with Pt-GG and Pt-AG intrastrand cross- Abstract links being the major lesions (1, 2). Oxaliplatin (cis-[(1R,2R)- Oxaliplatin (Eloxatin) isa third-generation platinum deriv- 1,2-cyclohexanediamine-N,N¶] oxalato (2-)-O,O¶ platinum; ative with an in vitro and in vivo spectrum of activity Eloxatin) is a third-generation platinum derivative bearing a distinct from that of cisplatin, especially in colon cancer 1,2-diaminocyclohexane carrier ligand and is now approved, cells. Here, we studied the molecular basis of this in combination with 5-fluorouracil, for the treatment of difference on the HCT-116 human colon carcinoma cell advanced colorectal cancer (3). In vitro comparisons of line (mismatch repair-deficient, wild-type functional p53). oxaliplatin with cisplatin showed that oxaliplatin requires Oxaliplatin inhibited HCT-116 cell proliferation with fewer DNA lesions than cisplatin to inhibit cell growth greater efficacy than cisplatin. At comparable concen- (1, 2, 4) and it has a broad potent antiproliferative activity, trations, cisplatin slowed down the replication phase and especially in cisplatin-resistant colon cancer cell lines (5, 6). activated the G2-M checkpoint, whereasoxaliplatin In line with this, preclinical studies using tumor models activated the G1-S checkpoint and completely blocked and clinical data showed that oxaliplatin is therapeutically the G2-M transition. With the aim of finding oxaliplatin- beneficial in cisplatin resistant tumors that are common specific target genes and mechanisms differing from in colorectal cancer (7, 8). Altogether, these data suggest those of cisplatin, we established the transcriptional that oxaliplatin might have different cellular targets and signatures of both products on HCT-116 cells using mechanisms of action compared with those of cisplatin, microarray technology. Based on hierarchical clustering, which have yet to be established. we found that (a) many more geneswere modulated by DNA damage resulting from exposure to cytotoxic agents oxaliplatin compared with cisplatin and (b) among the 117 induces cell cycle arrest, which mainly occurs at the G1-S modulated genes, 79 were regulated similarly by both or/and G2-M transition via activation of checkpoint drugs and, in sharp contrast, 38 genes were dose proteins that inhibit cyclin-dependent kinase (CDK) activ- dependently down-regulated by oxaliplatin and, conversely, ities (9). For example, inhibition of CDK4/cyclin D and/or up-regulated or unaffected by cisplatin. Interestingly, CDK2/cyclin E activity leads to G -phase arrest, inhibition several cell cycle–related genes encoding proteins in- 1 of CDK2/cyclin A activity delays S-phase progression, volved in DNA replication and G -M progression belong to 2 and the inhibition of CDK1/cyclin B, a key regulator of the thislatter group. RNA modulations,confirmed at the G -M transition, induces G arrest. CDK1 activation is protein level, were in accordance with oxaliplatin- and 2 2 controlled by dephosphorylation at Tyr15 by CDC25C cisplatin-induced cell cycle variations. Beyond the identi- phosphatase and CDC25C phosphatase is inactivated by fication of genesaffected by both drugs,the identified phosphorylation via Chk1 after DNA damage. Most studies oxaliplatin-specific target genes could be useful as on the effects of platinum drugs on cell cycle progression have been done with cisplatin and showed that it reduces the DNA synthesis rate (10, 11) with a subsequent S-phase slowdown (12) followed by G2-M arrest (12, 13). Although it has not yet been clearly established whether cisplatin- Received 6/27/05; revised 6/30/06; accepted 7/12/06. treated cells are arrested in the G2 or M phase, cisplatin- The costs of publication of this article were defrayed in part by the induced G -M arrest is consistent with the inhibition of the payment of page charges. This article must therefore be hereby marked 2 advertisement in accordance with 18 U.S.C. Section 1734 solely to CDK1/cyclin B activity by cisplatin (13). indicate this fact. The effects of oxaliplatin on the cell cycle have been Requests for reprints: Pierre Casellas, Sanofi-Synthelabo Recherche, reported similar to the effects of cisplatin. However, 371 rue du Professeur Joseph Blayac, F-34184 Montpellier Cedex 04, France. Phone: 33-4-67-10-62-90; Fax: 33-4-67-10-60-00. contrasting results on the effects of 1,2-diaminocyclohex- E-mail: [email protected] ane-platinum compounds on cell cycle progression have Copyright C 2006 American Association for Cancer Research. been reported (14). For example, on OVCA-429 ovarian doi:10.1158/1535-7163.MCT-05-0212 cancer cells, cisplatin arrests both wild-type p53 and

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mutant p53 cells in G2-M, whereas 1,2-diaminocyclohex- 12 hours and then counted using a Microbeta 1450 Trilux ane-acetato-Pt arrests wild-type p53 cells in G1 and mutant scintillation counter (Wallac, Wellesley, MA). Each experi- p53 cells in G2-M (14). mental point is done in quadruplicate. The proliferation In this context, further investigations on the effects of assay has been done thrice. oxaliplatin on cell cycle progression and on identification Cell Cycle Analysis of cell cycle–related target genes and mechanisms of action HCT-116 cells were plated (0.5 Â 106 per well) in six-well are essential. plates. Twenty-four hours later, cells were cultured for The aim of this study was to investigate the effects of 24 hours in the presence of drug or solvent. Cells were oxaliplatin on cell cycle progression and to identify cell harvested by trypsinization, washed twice with PBS, and cycle–related oxaliplatin-specific target genes and mech- resuspended in 70% ethanol in PBS and kept at 4jC anisms that would explain the higher efficacy of overnight. After washing, cells were incubated for 5 minutes oxaliplatin compared with cisplatin, especially in colon with PBS containing 0.25% Triton X-100, washed twice, carcinoma cells. To this aim, the effects of both resuspended in PBS-fetal bovine serum (1%), and labeled oxaliplatin and cisplatin were investigated on the HCT- with MPM-2 antibody (1 Ag final concentration; Upstate 116 colon carcinoma cell cycle. HCT-116 cells are Biotechnology, Charlottesville, VA) for 45 minutes at room deficient in DNA mismatch repair because of a genetic temperature. Cells were rinsed twice with PBS and incubated defect in the hMLH1 gene, which is located on chro- with Alexa Fluor 633–conjugated goat anti-mouse antibody mosome 3 (15). This deficiency is a common etiologic (10 Ag/mL final concentration; Molecular Probes, Eugene, factor for colon cancer (16) and, interestingly, is a key OR) for 30 minutes at room temperature. After washing factor for cisplatin and carboplatin resistance (6). HCT- twice with PBS, cells were resuspended in FITC-conjugated 116 cells express a functional p53 protein and have mouse IgGanti-cyclin B1 antibody solution (20 AL/test; BD mostly intact DNA damage–dependent checkpoints, thus PharMingen, San Diego, CA). Before analysis, cells were allowing cell cycle investigations. Furthermore, HCT-116 washed twice and stained with propidium iodide (PI) cells have already been used in analyses on the mecha- solution (5 Ag/mL PI in PBS containing 0.1% Triton X-100 nism of action of platinum compounds (6, 17). Until and 0.2 mg/mL RNase A). Samples were analyzed on a recently, the only key differentiating variable between Becton Dickinson (Heidelberg, Germany) FACScan, and the 1,2-diaminocyclohexane platinum and other platinum data were analyzed using CellQuest software. Each experi- compounds [which present a very different profile in mental point is done in duplicate and the results have the National Cancer Institute COMPARE program (6) and been confirmed at least in three independent experiments. a differential clinical activity profile], both as a single RNA Preparation agent and notably in combination (7) was mismatch For each collection point, the cell monolayer (15 Â 106 repair status (18). cells) was washed twice with PBS at 4jC and total RNAs Using a global gene expression approach, we examined were extracted using the RNeasy Midi kit (Qiagen, transcriptional modulations induced by both drugs and Valencia, CA) according to the manufacturer’s instructions. identified cell cycle–related genes that are inversely Microarray Assay regulated by oxaliplatin and cisplatin. RNA modulations RNA targets (biotin-labeled RNA fragments) were were confirmed at the protein level. These target genes that produced from 5 Ag total RNAs by first synthesizing we identified here may be important mediators of specific double-stranded cDNA followed by an in vitro transcrip- tumor cell response to oxaliplatin treatment. tion reaction and a fragmentation reaction. An hybridiza- tion mixture containing the cRNA fragments, probe array controls (Affymetrix, Inc., Santa Clara, CA), bovine serum Materials and Methods albumin, and herring sperm DNA was prepared and Cell Culture hybridized to human cancer G110 arrays, containing 1,700 The HCT-116 human colon cancer cell line was from the cancer-associated genes (Affymetrix) at 45jC for 16 hours. American Type Culture Collection (Rockville, MD). Cells The hybridized probe array was then washed. Bound were grown as monolayers in McCoy’s medium supple- biotin-labeled cRNA was detected with a streptavidin- mented with 10% FCS at 37jC in a humidified atmosphere phycoerythrin conjugate. Subsequent signal amplification containing 5% CO2. was done with a biotinylated anti-streptavidin antibody. Proliferation Assays The washing and staining procedures were automated HCT-116 cells were plated (5,000 per well) in 96-well using the Affymetrix fluidics station. Each human cancer Cytostar-T scintillating microplates (Amersham Pharmacia, G110 array was scanned once using a Hewlett-Packard Little Chalfont, United Kingdom) in the presence of (Palo Alto, CA) GeneArray scanner. Each RNA preparation oxaliplatin (supplied by Sanofi-Synthelabo Recherche, was hybridized to duplicate chips. Montpellier, France), cisplatin [commercially available from Microarray Data Analysis Sigma (St. Louis, MO) as a lyophilized powder in 50-mg Low-level data analysis was done using MAS version 5.0 vial], or solvent. Proliferation was measured by the software (Affymetrix.) by running absolute analyses on incorporation of [14C]thymidine (Amersham Pharmacia). each array and comparative analyses between pairs of arrays Cells were pulsed with [14C]thymidine (0.5 ACi/mL) for hybridized with control and treated cells, respectively.

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All arrays were scaled to a mean gene expression intensity of Immunofluorescence Staining 300. Detailed descriptions of the analysis algorithms have Cells were fixed in 70% ethanol for 15 minutes at been published (19, 20). Briefly, absolute analyses provide an 4jC and rinsed twice with PBS. Cells were then permea- estimate of the expression intensity (‘‘signal’’) of each gene bilized for 10 minutes at 4jC in PBS containing 0.25% as well as a variable, called ‘‘detection,’’ which determines Triton X-100, washed twice with PBS, and incubated with whether the gene can be considered as expressed (‘‘P’’ for PBS containing 1% bovine serum albumin. Primary anti- ‘‘present’’ and ‘‘M’’ for ‘‘marginal’’) or not (‘‘A’’ for bodies were added for 1 hour at room temperature. After ‘‘absent’’). Comparative analyses identify modulated genes washing twice with PBS, cells were incubated with Alexa (‘‘change’’ variable set at ‘‘I’’ or ‘‘MI’’ for up-regulation Fluor 633–conjugated goat anti-mouse antibody (10 Ag/mL and at ‘‘D’’ or ‘‘MD’’ for down-regulation associated with final concentration) for 30 minutes at room temperature. significant ‘‘change Ps’’). Comparative analyses also pro- Before analysis, cells were washed twice and stained for vide estimates of the expression intensity ratios for each 20 minutes at room temperature with PI solution (5 Ag/mL gene studied between the pairs of hybridized arrays being PI in PBS containing 0.1% Triton X-100 and 0.2 mg/mL compared. The ratios are expressed in base 2 logarithmic RNase A). After washing twice with PBS, slides were scale and are called ‘‘signal log ratios.’’ analyzed with a Zeiss (Thornwood, NY) LSM410 confocal For cluster analysis, only genes whose expression was laser scanning microscope. significantly modulated in at least one of the conditions studied were retained. The following criteria were used: expression modulations (as determined by the ‘‘change’’ Results variable) had to be reproduced in at least one comparative Cell Cycle Phase Distribution following Oxaliplatin or analysis, and the signal log ratios had to be above 1 in at CisplatinTreatment least one comparison. In addition, only genes whose To determine the effective drug concentrations, HCT-116 normalized expression intensity was at least 20 in one cells were treated with various concentrations of oxalipla- sample were considered. Finally, genes whose expression tin and cisplatin for 24 hours before a 12 h-thymidine was undetected (‘‘detection’’ equal to ‘‘A’’) in both treated pulse. In agreement with previously published data (6), and control samples in >75% of the comparisons were both drugs inhibited HCT-116 proliferation with oxalipla- excluded. Cluster analysis was done on signal log ratios tin more potent than cisplatin (i.e., its IC50 was 0.4 versus values weighed by the associated ‘‘change P.’’ Hierarchical 1.2 Amol/L for cisplatin). Cell growth inhibition by clustering was obtained by calculating similarity between oxaliplatin remained almost the same from 1 to 5 Amol/L genes as the Jaccard correlation and grouping following the (90% of inhibition), whereas the inhibition induced by average linkage algorithm (unweighted pair group method cisplatin only reached that high level at 5 Amol/L (data not with arithmetic mean) using the GeneMaths software shown). This indicated that oxaliplatin was f5-fold more package (Applied Maths, Inc., Austin, TX). potent in inhibiting DNA synthesis than was cisplatin. Western Blot Analysis Doses from 1 to 10 Amol/L of both drugs have been used to Cells (5 Â 106) were washed with ice-cold PBS and study the effects of cisplatin and oxaliplatin on the cell incubated with 750 AL cold lysis buffer (50 mmol/L Tris, 120 cycle and the comparison between the two drugs has been mmol/L NaCl, 0.5% NP40, protease inhibitor cocktail) for done with 1 Amol/L oxaliplatin and 5 Amol/L cisplatin. 10 minutes on ice. Whole-cell lysates were clarified by To investigate the effects of oxaliplatin and cisplatin on centrifugation at 4,000 rpm for 15 minutes at 4jC. Samples cell cycle progression, HCT-116 cells were treated for (50 Ag/condition) were heated in SDS sample buffer for 24 hours and analyzed by flow cytometry. The profiles 5 minutes at 95jC, run on 4% to 20% Tris-glycine gel (Novex, (Fig. 1A) show correlated dual-variable plots of cell count Invitrogen, Carlsbad, CA), and transferred to polyvinyli- versus PI uptake after 24 h of treatment with 1 and dene difluoride membranes (Amersham Biosciences, Little 5 Amol/L of each drug. They indicated that 1 Amol/L Chalfont, United Kingdom) using the Mini-Gel blot module oxaliplatin slightly increased the relative number of cells in (Invitrogen). Membranes were blocked for 1 hour at room the G2-M phase (28% versus 22% for control cells) and temperature with 5% bovine serum albumin in TBS [10 decreased the relative number of cells in the S phase (4% mmol/L Tris (pH 7.6) and 150 mmol/L NaCl] and then for 1 Amol/L oxaliplatin versus 20% for control cells), probed with anti-cyclin A (Neomarkers, Fremont, CA), anti- although it had little effect on the cell population in G1 cyclin B1 (Neomarkers), anti-CDK1 (Cell Signaling, Beverly, (68% for 1 Amol/L oxaliplatin versus 58% for control cells). MA), anti-phosphorylated CDK1 (Cell Signaling), anti- Under the same conditions, 5 Amol/L cisplatin increased A CDC25C (Cell Signaling), or anti-actin (Santa Cruz Biotech- the relative number of cells in G2-M (43% for 5 mol/L nology, Santa Cruz, CA) antibodies overnight at 4jC. cisplatin versus 22% for control cells) but, contrary to Incubation with the appropriate peroxidase-coupled sec- oxaliplatin, it increased the relative number of cells in the ondary antibody and subsequent detection by enhanced S phase (36% for 5 Amol/L cisplatin versus 20% for control chemiluminescence were done as recommended by the cells) and decreased the number of cells in G1 (19% for manufacturer (New England Biolabs, Ipswich, MA). Protein 5 Amol/L cisplatin versus 58% for control cells). levels were quantified using GeneTools analysis software G2 and M phases were further analyzed using the MPM- (Syngene, Inc., Frederick, MD). 2 labeling of mitotic cells. The MPM-2 antibody specifically

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Figure 1. Effects of 24 h of oxaliplatin and cisplatin (1 and 5 Amol/L) treatment on the cell cycle of asynchronously growing HCT-116 cells assessed by fluorescence-activated cell sorting analysis. A, correlated dual-variable plots of cell counts versus PI uptake. G1,S,andG2-M cell populations. B, correlated dual-variable plots of MPM-2 versus PI uptake. Green, mitotic cells; framed, cells in the S phase. C, immunofluorescence staining of the nuclear membrane after 24 h of oxaliplatin or cisplatin (1 and 5 Amol/L) treatment. After treatment, cells were stained with PI (red fluorescence) and lamin B2 antibody plus Alexa Fluor 633 – conjugated secondary antibody (green fluorescence) to visualize the nuclear integrity and nuclear membrane, respectively. Arrows, mitotic cells; asterisks, abnormal G1 cells.

recognizes epitopes on phosphoproteins expressed at an cisplatin and oxaliplatin showed that the effect of the drugs early stage of mitosis (21). Correlated dot plots of MPM-2 on cell distribution was concentration dependent from 1 to labeling versus PI uptake are shown in Fig. 1B. Both 5 Amol/L drug concentration and plateaued at 5 Amol/L in 5 Amol/L cisplatin and 1 Amol/L oxaliplatin blocked cells both cases (data not shown). in the G2 phase, thus reducing the number of mitotic cells Taken together, these data showed that cisplatin slowed compared with control cells. With cisplatin (10 Amol/L) down the replication phase and partially blocked cells in treatment, few cells still bypassed the G2 checkpoint and the G2 phase, probably allowing aberrant mitosis. More entered mitosis (data not shown), whereas oxaliplatin interestingly, they showed that oxaliplatin strongly A (5 and 10 mol/L)–treated cells did not evade the G2 blocked cells in the G1 and G2 phases. Although they are checkpoint (Fig. 1B; data not shown). To further document close compounds, cisplatin and oxaliplatin did not alter the effects of oxaliplatin and cisplatin on the M-phase cell cycle progression to the same extent, which suggests entry, we assessed the nuclear membrane integrity, as its that oxaliplatin and cisplatin may have common and breakdown is a common feature of mitotic cells. The results distinct mechanisms of action. of fluorescence microscopy experiments with PI labeling Oxaliplatin and Cisplatin Gene Expression Profiles (DNA labeling) and lamin B2 antibody staining (nuclear A global gene expression approach was used to identify membrane labeling), shown in Fig. 1C, confirmed the cisplatin- and oxaliplatin-specific target genes. Using DNA reduction of the number of mitotic cells following 24 hours chips containing 1,700 human cancer-related genes, we of treatment with 1 Amol/L oxaliplatin or 5 Amol/L identified genes whose expression was dose dependently cisplatin compared with the control. Abnormal G1 cells affected by oxaliplatin and cisplatin after 48 hours of were observed among the cisplatin-treated cells (Fig. 1C). treatment (shorter treatments did not give significant Their shapes suggested that they were G1 cells issued from results). We compared gene expression profiles of HCT- abnormal mitosis. Experiments done with 1 to 10 Amol/L 116 cells treated for 48 hours with oxaliplatin and cisplatin

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concentrations ranging from 0.5 to 10 Amol/L. Below catenin a1. Histone H1 is necessary for the condensation of 0.5 Amol/L drug, we did not detect any RNA modulations. nucleosome chains into higher order structures; retino- Gene expression profiles were analyzed using hierarchical blastoma 1, a nuclear protein, is a potent inhibitor of E2F- clustering (Fig. 2). Of these genes, 117 were significantly mediated transactivation; and catenin a1 associates with modulated (see Materials and Methods for the selection the cytoplasmic domain of a variety of cadherins. Subclus- criteria). Modulated genes can be subgrouped within three ter C revealed that cisplatin and oxaliplatin induced subclusters: (A) 37 genes dose dependently up-regulated distinct gene modulation patterns. It contained genes by both drugs, (B) 42 genes dose-dependently down- strongly down-regulated by oxaliplatin, whereas they were regulated by both drugs, (C) and 38 drug-specific gene either up-regulated or unaffected by cisplatin. What really modulations. Tables 1, 2 and 3 show the major significantly matters is that the pattern of inverse response remained modulated genes from subclusters A, B, and C, respective- valid for the 1 Amol/L oxaliplatin versus 5 Amol/L cisplatin ly, and their functional assignments. Subcluster A comparison (Fig. 2, framed). From this subcluster C, some contained proapoptotic genes, such as Bax, Bak, and Fas/ cell cycle–related genes were involved in the G2-M Apo-1. Among these genes, oxaliplatin-induced modula- transition (i.e., cyclin B, CDC25C, and CDK1) or in the tions of Bax and Bak have already been reported (17). duplication phase (i.e., CDC6, cyclin A, replication protein A, This subcluster also contained the WAF-1/p21 gene, which replication factor C,andprimase). Ki-67, the nucleolar is involved in cell cycle progression and has already been proliferation marker, also belonged to subcluster C, such described as up-regulated in response to cisplatin treatment as B-Myb, another nuclear protein involved in cell (22). Subcluster B included histone H1, retinoblastoma 1, and proliferation. Several genes involved in the DNA replication (i.e., P1-Cdc21, Cdc7-related kinase, and thymidine kinase) were also inversely regulated by cisplatin and oxaliplatin. In addition, we observed modulation of centromere protein A and nucleosome assembly protein 1. Centromere protein A is a histone H3-like protein that is an essential component of centromeres and specifies the mitotic behavior of chromosomes, and the nucleosome assembly protein 1, a histone chaperone that mediates assembly proteins, participates in DNA replication and contributes to regulation of cell proliferation. We have tested two more cell lines: the HT-29 human colon cell line and the A2780 human ovary cell line. In the HT-29 cell line (data not shown), the genes from subcluster C, specific of the oxaliplatin transcriptional signature in the HCT-116 cell line, were unchanged or up-regulated by both cisplatin and oxaliplatin. No oxaliplatin-specific transcriptional signature appeared with this cell line. In the A2780 cell line (data not shown), the genes from subcluster C were down-regulated by oxaliplatin (0.5–10 Amol/L) as in the HCT-116 cell line. Protein Expression in HCT-116 Cells following Oxali- platin or CisplatinTreatment Cell cycle–related gene modulations were further investigated at the protein level. As shown in Fig. 3A and Table 4, the protein levels were correlated with the mRNA modulations: the cyclin A, cyclin B, CDK1, and CDC25C protein expressions were unchanged or slightly repressed by 24 hours of oxaliplatin (1 Amol/L) treatment, strongly repressed after 48 hours of oxaliplatin (1 Amol/L) treatment, and either unaffected or slightly enhanced by 24 and 48 hours of cisplatin (5 Amol/L) treatment. As CDK1 activity is regulated by phosphorylation, we used an Figure 2. Hierarchical clustering of gene expression data measured after antibody targeting the CDK1 phosphorylated form (Tyr15). 48 h of oxaliplatin and cisplatin treatment. The single-dimension clustered gene expression measured from G110 array analyses using Jaccard The results showed that expression of the phosphorylated correlation and algorithmunweighted pair group methodwith arithmetic form was down-regulated by 48 hours of oxaliplatin mean. Row, a separate gene; column, a treated sample hybridized onto an treatment (P-CDK1/actin ratio of 0.16 for 1 Amol/L array. Green scale, down-regulation; red scale, up-regulation; white oxaliplatin; Table 4) and slightly up-regulated by 48 hours scale, no change. Data are relative to untreated cells. Right, subclusters A, B, and C. Underlined, comparison of 1 Amol/L oxaliplatin versus of cisplatin treatment (P-CDK1/actin ratio of 1.59 for 5 Amol/L cisplatin. 5 Amol/L cisplatin; Table 4). As cyclin B is one of the key

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Table 1. Functional assignment of major genes that are induced several cytotoxic agents, including antimetabolite agents M by 0.5 to 10 mol/L cisplatin and oxaliplatin (subcluster A) (methothrexate and 5-fluorouracil), alkylating agents (melphalan, oxaliplatin, and cisplatin), and topoisomerase Function Gene name Accession no. inhibitors (camptothecin, doxorubicin, and etoposide). Apoptosis Bax a 2065_s_at Hierarchical clustering of anticancer drugs showed that Bax d 1997_s_at cisplatin and oxaliplatin were grouped together, close to Bax c 2067_f_at the alkylating agent melphalan (data not shown). This was Bak 846_s_at in accordance with the fact that cisplatin and oxaliplatin Fas/Apo-1 1440_s_at belong to the same drug family and are also alkylating MDM-2 1880_at agents. As expected, the signatures of cisplatin and Cell cycle p21waf1/cip1 2031_s_at oxaliplatin had common features (Fig. 2). Modulations of Cyclin G1 1920_s_at genes involved in the apoptosis response, such as Bax and MDM-2 1880_at Fas, are part of this platinum signature, indicating a Proliferating cell nuclear antigen 1824_s_at common cell death mechanism. The hierarchical clustering Transcription RNA polymerase II 503_at Signal Ras 1590_s_at elicited a cluster of 38 genes that could help to differentiate transduction the effects of oxaliplatin and cisplatin as they are Annexin II 757_at specifically modulated by cisplatin or oxaliplatin. Cell Inositol polyphosphate 1-phosphatase 656_at cycle–related genes are included in this cluster. Previous Inositol polyphosphate 5-phosphatase 172_at studies showed that these cell cycle–related genes share DNA replication Proliferating cell nuclear antigen 1824_s_at common sequences in their promoters: the CDE and CHR Drug resistance Semaphorin E 377_g_at promoter elements (26). Moreover, these sequences have already been described as common regulators of these genes and involved in G2-M arrest (26). Considering that proteins in the G2-M transition, we measured its expression this sequence is very rich in GC bases, altogether these data by flow cytometry (Fig. 3B). Figure 3B shows a significant highly suggested that this promoter sequence has a key decrease in cyclin B expression following treatment with role in oxaliplatin-specific transcriptional signature and 1 Amol/L oxaliplatin compared with untreated cells. By that these genes may be modulated by oxaliplatin. contrast, in the same conditions, 5 Amol/L cisplatin The transcriptional study results and their validation at increased cyclin B expression of cells in the G2-M phase. the protein level suggested that two different mechanisms These results are visible after 24 hours of treatment but are responsible for oxaliplatin- and cisplatin-induced G2 stronger after 48 hours. transition control. Oxaliplatin induced a strong inhibition of CDK1 and cyclin B protein expression. It is now widely Discussion accepted that the G2-M transition is partly governed by the It is widely accepted that DNA damage is largely CDK1-cyclin B complex. G2 arrest associated with cyclin B responsible for the cytotoxic properties of platinum repression has already been reported on treatment with (23, 24). Interestingly, oxaliplatin, which is more cytotoxic adriamycin, etoposide, and irinotecan (27), suggesting than cisplatin (6), forms fewer DNA adducts than cisplatin that cyclin B repression is involved in the G2 arrest does [e.g., 0.86 F 0.04 versus 1.36 F 0.01 adducts/106 bp/ mechanism induced by anticancer drugs. p21waf1/cip1 exerts A 10 mol/L drug/1 hour in CEM cells (4); refs. 1, 2, 4]. This a key role in controlling the G2 arrest by inhibiting the suggests different mechanisms of action. CDK1-cyclin B complex. Microarray analysis showed that, Activation of cell cycle checkpoints is a general cellular under oxaliplatin treatment, p21waf1/cip1 RNA expression response following exposure to cytotoxic agents. These was up-regulated by 4.8-fold (only 1.6-fold activation with checkpoints arrest cells at the G1-S or G2-M transitions. Previous studies indicated that cisplatin and other plati- Table 2. Functional assignment of major genes that are num agents predominantly reduce the rate of DNA repressed by 0.5 to 10 Mmol/L cisplatin and oxaliplatin synthesis (10, 12) and inhibit cell cycle progression at the (subcluster B) G1-S and/or G2-M phases (12, 25). Here, we show, in agreement with previously published data (10, 12, 25), that Function Gene name Accession no. cisplatin slowed down cells in the S phase and disturbed the G2-M transition. More interestingly, our results Cell cycle CDC25B 1347_at indicated that in HCT-116 cells, oxaliplatin strongly CDKN2A 1713_s_at CDKN1C 1787_at blocked cells in the G1 and G2 phases. The fact that Retinoblastoma 1 1937_at cisplatin and oxaliplatin had different effects on cell cycle Transcription Transcription elongation factor 1073_at progression suggests that the two drugs do not have the Signal transduction Sky 2086_s_at same mechanism of action. HER-3 1585_at In this study, we analyzed the effects of cisplatin and Cell adhesion Catenin a1 2085_s_at oxaliplatin at the gene expression level. We have an Nucleosome assembly Histone H1 580_at anticancer drug transcriptional signature database with

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cisplatin treatment) as early as 6 hours of treatment (data Table 3. Functional assignment of major genes that are induced M M not shown). We hypothesize that the p21waf1/cip1 pathway by 0.5 to 10 mol/L cisplatin and repressed by 0.5 to 10 mol/L oxaliplatin (subcluster C) may be involved in early events of the oxaliplatin-induced G2 block. Absence of cyclin B and CDK1 after 48 hours of Function Gene name Accession no. oxaliplatin treatment may be involved as late events in the establishment of a very strong arrest of cells in G2 phase by Cell cycle Cyclin A 1943_at oxaliplatin. Cisplatin treatment did not decrease the CDK1 Cyclin B 1945_at or cyclin B protein level, rather cisplatin-induced G2 arrest CDK1 1803_at was correlated with a slight accumulation of both proteins. CDC25C 1584_at It has been widely shown that the phosphorylation of mki67 418_at CDK1 on Tyr15 by CDC25C (28) inactivates the CDK1- B-myb 1854_at cyclin B complex, which blocks M-phase entry. Considering CIP2 1599_at Mad2 1721_g_at the total amount of CDK1 (P-CDK1 and CDK1) in our DNA replication Replication factor C 1053_at study, the CDK1 phosphorylated form was not increased DNA primase 798_at by cisplatin, suggesting that the CDK1 activity is not Replication protein A 527_at responsible for the G2 blockage induced by cisplatin. Cdc7-related kinase 1809_at The results of the gene and protein expression analysis P1-Cdc21 981_at also enabled us to gain insight into the mechanism of Thymidine kinase 910_at action involved in the oxaliplatin-induced G1 checkpoint. It NAP1 571_at is well known that the inhibition of cyclin A/CDK2 activity CDC6-related protein 1536_at delays or prevents S-phase progression. In our experi- Nucleosome assembly Centromere protein-A 527_at ments, the expression of cyclin A and CDK2 RNAs drastically decreased only after 48 hours of treatment, suggesting that inhibition of cyclin A and CDK2 RNA expression may be a consequence of the G1 arrest. The cancers, and this is seriously in cancers with known absence of cyclin A and CDK1 proteins after 48 hours of prevalent somatic or epigenetic mismatch repair deficiency oxaliplatin treatment could contribute to maintain the G1 (e.g., colon, ovarian, or endometrial cancers). Interestingly, arrest. Oxaliplatin also down-regulated genes encoding in such tumors, mismatch repair-deficient phenotype is proteins involved in the initiation of DNA replication, increasingly prevalent with tumor progression, although such as CDC6 and primase, or in the progression of the prospective clinical translational work has never been DNA replication process, such as replication protein A and implemented to completely validate this insight (7). replication factor C, and this may also contribute to late An intriguing and positive characteristic of oxaliplatin is blockage at the S phase entry. its marked preclinical and clinical synergy with a wide A checkpoint response induced by cytotoxic agents can variety of anticancer agents, mainly antimetabolites, such have both positive and negative effects on apoptosis (9, 29). as fluorouracil (32) or gemcitabine (33), but also with It has been shown that disruption of the G2 checkpoint cisplatin (6). In most cases, these oxaliplatin-based combi- control response elicited by cytotoxic agents often increases nations are superior to cisplatin-based combinations. This the cellular sensitivity to these drugs (9, 29, 30) because superiority is seen above and beyond the mismatch repair unbridled cell cycle progression in the presence of DNA prevalence as it is the case with taxanes (34) or Vinca damage is usually lethal. For this reason, components of alkaloids (35). Preliminary work suggests that DNA synthe- the G2 checkpoint response are potential targets for new sis (via the thymidylate synthase) and repair mecha- chemotherapeutic agents. The relationship between the nisms (via the excision repair cross complementing 1) are checkpoint response of the other phases of the cell cycle also implicated in the synergy observed with 5-fluorouracil and the induction of apoptosis is less established (9, 29). It (36). The current data have been obtained in a wild-type has been shown that G1 checkpoint abrogation decreases functional p53 cell line. Our study has been extended to the drug sensitivity (31). This was supported by our results p53-proficient A2780 cell line and the p53-deficient HT-29 that showed that the G1 checkpoint response was associ- cell line. Our results on the p53-proficient A2780 cell line ated with increased sensitivity. Further studies should be confirmed the oxaliplatin-specific transcriptional signature. carried out to examine whether the effect of oxaliplatin on Our results also indicated that there was no oxaliplatin- the G1 checkpoint is linked with the ability of oxaliplatin to specific transcriptional signature with the p53-deficient HT- bypass cisplatin resistance and/or how does it may 29 cell line. These data support the fact that the G2 cell cycle increase the apoptotic consequences of its combination arrest associated with a decrease in cyclin A and B level with other agents. is p53 dependent (26). The G2 checkpoint activity seems The greater efficacy of oxaliplatin compared with to be a new and major differentiating factor between cisplatin (i.e., isocytotoxicity with fewer DNA/platinum 1,2-diaminocyclohexane platinums and the other DNA adducts) does not always translate into clinical settings. alkylators, and it is probably linked to the nonrepairability Indeed, oxaliplatin at recommended doses shows only of transcriptional gene sites associated with the G2-M- marginal clinical activity as a single agent in most human related cyclins cascade. It could also explain the enhanced

Mol Cancer Ther 2006;5(9). September 2006

Figure 3. Protein expression of cell cycle – related genes. A, Western- blotting. Cyclin B, cyclin A, CDK1, P-CDK1, CDC25C, and actin protein levels in HCT-116 cells after 24 and 48 h of treatment with 1 and 5 Amol/L oxaliplatin or cisplatin and control (C). B, flow cytometry. Correlated dual- variable plots of cyclin B versus PI uptake. HCT-116 cells were treated for 24 and 48 h with 1 and 5 Amol/L oxaliplatin and cisplatin.

drug sensitivity to other anticancer agents acting on DNA technology, we established the oxaliplatin and cisplatin or other targets when combined with oxaliplatin, such as transcriptional signatures in the HCT-116 human colon 5-fluorouracil, gemcitabine, taxanes, etc., which act through carcinoma cell line. We noted a platinum transcriptional cell cycle arrests at the G1-S and/or M phases. signature and, in parallel and for the first time, observed In summary, we showed for the first time that cells cell cycle–related genes that were conversely regulated by treated with cisplatin and oxaliplatin respond differently oxaliplatin and cisplatin. These modulations were con- with regard to the genes that are modulated, suggesting firmed at the protein level. These results highly suggest two different mechanisms of action. We also showed that that the cell cycle modifications induced by cisplatin and oxaliplatin was a strong and specific cyclin cascade G2-M oxaliplatin are the result of distinct cellular mechanisms. transition blocker and, to our knowledge, the first agent Finally, the oxaliplatin-specific target genes that have been with such blocking specificity. Using a DNA array identified give clues for the mechanism of action of

Table 4. Protein/actin ratio (percentage of treated cells versus nontreated cells)

Treatment (24 hours) Treatment (48 hours)

Cisplatin Oxaliplatin Cisplatin Oxaliplatin

1 Amol/L 5 Amol/L 1 Amol/L 5 Amol/L 1 Amol/L 5 Amol/L 1 Amol/L 5 Amol/L

Cyclin B 1.02 1.52 0.91 0.56 0.80 1.41 0.05 0.02 Cyclin A 1.03 0.97 0.78 0.48 0.67 1.15 0.16 0.03 CDK1 0.98 1.20 0.90 1.19 1.18 2.20 0.77 0.43 P-CDK1 0.92 1.21 1.09 0.75 0.89 1.59 0.16 0.02 CDC25C 1.53 0.91 1.11 0.66 2.05 2.35 0.61 0.38

Mol Cancer Ther 2006;5(9). September 2006

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